Ca 2+/calmodulin-dependent protein kinase kinase-β acts upstream of AMP-activated protein kinase in mammalian cells

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Ca 2+/calmodulin-dependent protein kinase kinase-β acts upstream of AMP-activated protein kinase in mammalian cells

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  Ca 2+ /calmodulin-dependent protein kinase kinase  β phosphorylation of Sirtuin 1 in endotheliumis atheroprotective Liang Wen a,b , Zhen Chen b , Fan Zhang c , Xiaopei Cui b , Wei Sun b , Greg G. Geary d , Yinsheng Wang c , David A. Johnson b ,Yi Zhu a,1 , Shu Chien e,1 , and John Y.-J. Shyy b,f,1 a Department of Physiology and Pathophysiology, Peking University Health Sciences Center, Beijing 100191, China;  b Division of Biomedical Sciencesand  c Department of Chemistry, University of California, Riverside, CA 92521;  d Department of Kinesiology and Health Sciences, California State University,San Bernardino, CA 92407; and Departments of  e Bioengineering and  f Medicine, University of California, San Diego, La Jolla, CA 92093Contributed by Shu Chien, May 16, 2013 (sent for review March 3, 2013) Atheroprotective  󿬂 ow exerts antioxidative and anti-in 󿬂 ammatoryeffects on vascular endothelial cells (ECs), in part through theinductionofSirtuin 1 (SIRT1),a class III histone deacetylase. The roleof Ca 2+ /calmodulin-dependent protein kinase kinase (CaMKK) β  in 󿬂 ow induction of SIRT1 both in vitro and in vivo was investigated.Pulsatile shear stress mimicking atheroprotective  󿬂 ow increasedthe level of SIRT1 in cultured ECs by enhancing its stability, and thiseffect was abolished by inhibition or knockdown of CaMKK β . Flow-enhanced SIRT1 stability was primarily mediated by CaMKK β  phos-phorylation of SIRT1 at Ser-27 and Ser-47, as evidenced by in vitrokinase assay, mass spectrometry, and experiments using loss- orgain-of-function SIRT1 mutants. Flow-induced CaMKK β  phosphory-lation of SIRT1 Ser-27 and Ser-47 increased antioxidative and anti-in 󿬂 ammatory capacities. Ablation of CaMKK β  or SIRT1 in micewith an apolipoprotein E-null background showed increased ath-erosclerosis both in athero-prone and in athero-protective areas.The results suggest that the CaMKK β -SIRT1 axis in ECs is mechano-sensitive, antioxidative, and anti-in 󿬂 ammatory. S hear stress-imposed endothelial responses affect vascularphysiology and pathophysiology. Ample evidence indicatesthat atheroprotective 󿬂 ow with pulsatile shear stress (PS) modulatesendothelial homeostasis via antioxidative and anti-in 󿬂 ammatory effects onvascular endothelialcells(ECs).Thesebene 󿬁 cialeffectsare mediated in part by the induction of Sirtuin (SIRT)1 (1).Functioning as a class III histone deacetylase, SIRT1 modulatescellular functions through deacetylation of targets such as Fork-head box (Fox)O, peroxisome proliferator-activated receptor(PPAR) γ ,PPAR γ coactivator(PGC)1 α ,andnuclearfactorkappa-light chain enhancer of activated B cells (NF- κ B), which areinvolved in oxidative and in 󿬂 ammatory states of the cell (2).SIRT1 ablation in ECs blocks angiogenesis in vitro and in vivo (3),and transgenic mice with EC-speci 󿬁 c SIRT1 overexpression showdecreased atherosclerosis (4). Additionally, SIRT1 can deacety-late endothelial nitric oxide synthase (eNOS) to stimulate eNOSactivity and increase NO production (5).The expression of SIRT1 is controlled at multiple levels, in-cluding transcription, posttranscription, and posttranslation (6).Posttranslationally, the SIRT1 level and/or its activity can bemodulated by sumoylation and phosphorylation (7, 8). Also, themRNA stability of SIRT1 can be regulated by microRNAs(9, 10). More than 10 residues of SIRT1 are phosphorylated/ dephosphorylated in multiple cell types under various conditions(7).Amongthese,Ser-27andSer-47inhumanSIRT1arethemoststudied. Several kinases, including c-Jun N-terminal kinase(JNK) 1, JNK2, mammalian target of rapamycin, and cyclin-dependent kinase 5 (CDK5), are implicated in the phosphoryla-tion of Ser-27 and Ser-47 (11-14), but the functional consequenceof their phosphorylation is unclear. None of the identi 󿬁 ed kinasesinvolved in SIRT1 phosphorylation has been shown to respond toatheroprotective  󿬂 ow. Given the link between shear stress andSIRT1, there could be a  󿬂 ow-responsive cytoplasmic kinase thatphosphorylates SIRT1 to enhance its activity.Like SIRT1, AMP-activated protein kinase (AMPK) functionsas a master regulator in stress response and energy homeostasis ineukaryotic cells. SIRT1 and AMPK are coregulated by caloricrestriction, resveratrol, and exercise (15, 16), which coordinately deacetylate and phosphorylate a common set of molecular targetssuch as PGC1 α  (17). Because SIRT1 and AMPK are regulated inconcert, a common upstream regulator is likely to exist. Consid-ering that elevated AMPK activity requires the phosphorylationof Thr-172 in its  α  subunit by AMPK kinases (AMPKKs), an AMPKKmightalsophosphorylateSIRT1toregulateitsactivityorexpression. The probable kinases involved in such regulation areCa 2+  /calmodulin-dependent protein kinase kinase (CaMKK) β and liver kinase B1 (LKB1), the most-studied AMPKKs.Here, through an examination of the mechanism by whichSIRT1 level and activity are enhanced by atheroprotective PS inECs, we report that CaMKK  β  phosphorylates SIRT1 at Ser-27 andSer-47 to increase its stability and activity. The translational signif-icance of these  󿬁 ndings is demonstrated by the increased athero-sclerotic lesions in mouse lines with ablation of CaMKK  β  or SIRT1. Results Shear Stress-Induced SIRT1 Depends on CaMKK β .  To establish a link between CaMKK  β  and SIRT1 in the context of endothelium ex-posed to atheroprotective  󿬂 ow, we  󿬁 rst examined CaMKK  β  acti- vation and its correlation with SIRT1 level in human umbilical Signi 󿬁 cance Different  󿬂 ow patterns in the arterial tree determine the se-verity and topographic distribution of atherosclerosis. Athero-protective  󿬂 ow exerts anti-in 󿬂 ammatory and antioxidativeeffects on vascular endothelial cells, and the underlying mech-anism involves the 󿬂 ow-induced upregulation of Sirtuin (SIRT)1.This study reveals that athero-protective 󿬂 ow activates Ca 2+ /calmodulin-dependent protein kinase kinase (CaMKK) β , which,in turn, phosphorylates SIRT1 at Ser-27 and Ser-47 to increasethe stability and activity of SIRT1. The mechanosensitiveCaMKK β -SIRT1 pathway alleviates in 󿬂 ammatory and redoxstatus in theendothelium.Suchaconclusion isevidentfromthedrastically increased atherosclerosis in mice lacking CaMKK β  orendothelial SIRT1. Author contributions: L.W., Z.C., D.A.J., Y.Z., S.C., and J.Y.-J.S. designed research; L.W., Z.C.,F.Z., X.C., W.S., G.G.G., and Y.W. performed research; L.W., Y.Z., and J.Y.-J.S. analyzed data;and L.W., D.A.J., Y.Z., S.C., and J.Y.-J.S. wrote the paper.The authors declare no con 󿬂 ict of interest. 1 To whom correspondence may be addressed. E-mail: yizhuucr@yahoo.com, shuchien@ ucsd.edu, or jshyy@ucsd.edu. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1309354110/-/DCSupplemental. E2420 – E2427  |  PNAS  |  Published online June 10, 2013  www.pnas.org/cgi/doi/10.1073/pnas.1309354110   vein endothelial cells (HUVECs) subjected to PS. As illustratedin Fig. 1  A , both CaMKK  β  (the upper band recognized by ananti – pan-CaMKK) and SIRT1 levels were signi 󿬁 cantly elevated at 4and 8 h after PS. As expected, there was an increase in phosphory-lation of CaMKK  β  targets [Ca 2+  /calmodulin-dependent proteinkinase (CaMK)I Thr-177 and AMPK Thr-172] and AMPK target[acetyl-CoAcarboxylase(ACC)Ser79].Furthermore,theincreasedSIRT1 level was consistent with the decreased acetylation of FoxO1, a SIRT1 target. To test whether CaMKK  β  mediates PSinduction of SIRT1 and AMPK, CaMKK  β  was knocked down by CaMKK  β  siRNA or inhibited by STO-609, a CaMKK-speci 󿬁 cinhibitor. Such CaMKK  β  suppressions blocked the PS-inducedincreases in the levels of SIRT1 and AMPK phosphorylation(Fig. 1  B and C ). The shear stress-dependentregulationofSIRT1and AMPK was also abolished in CaMKK  β −  /  − murine embry-onic  󿬁 broblasts (MEFs), compared with control CaMKK  β +/+ Fig. 1.  CaMKK β isrequiredforSIRT1inductioninECsunderPS.(  A – C  )ImmunoblotsofHUVECssubjectedtoPSfortheindicatedtimeperiods(  A ),pretreatedwithSTO-609(2.5  μ g/mL)orDMSO for30minandthensubjectedtoPSfor8h( B ),andtransfectedwithcontrolsiRNAorCaMKK β  siRNAandthenexposedtoPSfor8horkeptunder static conditions for the same time ( C  ). ( D ) Immunoblots of CaMKK β +/+ and CaMKK β −  /  − MEFs exposed to static condition or PS for 8 h. The antibodies againsttargeted proteins are indicated for each immunoblot. Bar graphs below immunoblots summarize the means ± SEM of three independent experiments. * P  < 0.05. Fig. 2.  PS increases SIRT1 stability via CaMKK β  phosphorylation at Ser-27 and Ser-47. Immunoblots of HUVECs pretreated with CHX (0.1 mg/mL) for 30 minbefore PS or kept under static conditions for the indicated time (  A  and  B ), transfected with or without control siRNA or CaMKK β  siRNA and then PS for 8 h ( B and  C  ), and transfected with control siRNA or CaMKK β  siRNA and then PS or static conditions for 8 h ( D ). ( E  ) Immunoblots of lysed SIRT1 −  /  − MEFs transfectedwith plasmid encoding SIRT1-S27AS47A or SIRT1-S27DS47D and then treated with CHX for the indicated times. The antibodies against targeted proteins areindicated. The plots below the immunoblots summarize the mean  ±  SEM results from three independent experiments. * P   <  0.05. Wen et al. PNAS  |  Published online June 10, 2013  |  E2421       P      H      Y      S      I      O      L      O      G      Y      P      N      A      S      P      L      U      S  MEFs (Fig. 1  D ). These results indicate that CaMKK  β  playsa critical role in mediating the PS regulation of SIRT1. CaMKK β  Phosphorylation of SIRT1 Enhances SIRT1 Stability.  We theninvestigated the molecular basis by which PS increases the level of SIRT1. Because the mRNA level of SIRT1 did not change in ECs with the applied PS (Fig. S1  A ), we examined whether the elevatedSIRT1 level was attributable to increased translation or proteinstability. With translation inhibited by cycloheximide (CHX), thedegradation rate of SIRT1 in ECs was signi 󿬁 cantly greater understatic conditions than under PS (Fig. 2  A ), suggesting an increasedSIRT1 stability under PS. This  󿬂 ow-associated effect was abol-ished with CaMKK  β  knockdown (Fig. 2  B ). Because SIRT1 sta-bility was reported to be affected by phosphorylation of its Ser-27and Ser-47 residues (12), we investigated whether PS-increasedSIRT1 stability was mediated via phosphorylation of SIRT1 atSer-27 and Ser-47 and, if so, whether these modi 󿬁 cations requireCaMKK  β . As shown in Fig. 2 C , PS increased the phosphorylationof SIRT1 Ser-27 and Ser-47, which was concomitant with increasedSIRT1 level. Moreover, CaMKK  β  siRNA knockdown greatly at-tenuated the phosphorylation of Ser-27 and Ser-47, both at thebasal level and under PS (Fig. 2  D ). To further examine the role of Ser-27 and Ser-47 phosphorylation on SIRT1 stability, we overex-pressed the phosphomimetic (i.e., SIRT1-S27D47D) or dephos-phomimetic (i.e., SIRT1-S27AS47A) mutant in SIRT1 −  /  − MEFsfor CHX pulse-chase experiment. As shown in Fig. 2  E , the exog-enously expressed SIRT1-S27DS47D was more stable than SIRT1-S27AS47A.Takentogether,theseresultsindicatethatPSincreased Fig. 3.  CaMKK β  phosphorylates SIRT1 at Ser-27 and Ser-47. (  A ) Alignment of peptide sequences  󿬂 anking SIRT1 Ser-27, SIRT1 Ser-47, AMPK Thr-172, CaMKIThr-177, CaMKIV Thr-196, PKB Thr-308, and BRSK1 Thr-189. ( B  and  C  ) Immunoblots of reaction products of mixtures of recombinant GST-CaMKK β  (50 ng)incubated with recombinant human SIRT1 (200 ng) ( B ) or AMPK (200 ng) ( C  ) with 2 mM Ca 2+ and recombinant calmodulin for 12 h ( B ) or for 30 min ( C  ) at30 °C. The phosphorylation of SIRT1 and AMPK was determined with the indicated antibodies. ( D  and  E  ) MS/MS of phosphorylated SIRT1 tryptic peptidescorresponding to residues 23 – 34 (EAASSPAGEPLR) ( D ) and 47 – 58 (SPGEPGGAAPER) ( E  ) obtained from the kinase reaction mixtures described in  B  and ana-lyzed by LC-MS/MS. The asterisk indicates that an ion bears a phosphate group, and neutral loss of an H 3 PO 4  is represented by  Δ . E2422  |  www.pnas.org/cgi/doi/10.1073/pnas.1309354110 Wen et al.  SIRT1stabilityandthatthiswasassociatedwithphosphorylationof SIRT1 at Ser-27 and Ser-47 and dependent upon CaMKK  β . CaMKK β  Phosphorylates SIRT1 at Ser-27 and Ser-47.  CaMKK  β phosphorylates AMPK at Thr-172, CaMKI at Thr-177, Ca 2+  / calmodulin-dependent protein IV (CaMKIV) at Thr-196, proteinkinase B (PKB) at Thr-308, and BR serine/threonine kinase(BRSK)1 at Thr-189 (18-22). Homology among amino acidsequences adjacent to SIRT1 Ser-27, Ser-47, and the respectiveCaMKK  β  phosphorylation sites of AMPK, CaMKI, CaMKIV,PKB, and BRSK1 (Fig. 3  A ) indicates that CaMKK  β  could bea SIRT1 kinase. To verify this, we  󿬁 rst performed in vitro kinaseassay using recombinant SIRT1 and CaMKK  β . In the presence of Ca 2+  /CaM, SIRT1 phosphorylation at both Ser-27 and Ser-47 wasincreased (Fig. 3  B ). As a positive control, AMPK phosphorylationat Thr-172 was increased as well (Fig. 3 C ). Additionally, nano-liquid chromatography/tandemmass spectrometry (nano – LC-MS/ MS) analysis con 󿬁 rmed that CaMKK  β  can phosphorylate Ser-27and Ser-47. The phosphorylation of SIRT1 at Ser-27 and Ser-47 within the corresponding tryptic peptides was veri 󿬁 ed by charac-teristic neutral loss of a phosphoric acid group (98 Da) (Fig. 3  D and  E ). In addition, the sites of phosphorylation were supportedby characteristic sequence ions (y and b ions) observed in theMS/MS (Tables S1 and S2). Thus, CaMKK  β  can directly phos-phorylate SIRT1 at Ser-27 and Ser-47. PS Up-Regulates Antioxidative and Anti-in 󿬂 ammatory Genes viaCaMKK β -SIRT1.  Like SIRT1, PS exerts antioxidative and anti-in 󿬂 ammatory effects on ECs, in part, by up-regulation of anti-oxidative and anti-in 󿬂 ammatory genes (2, 23, 24). In our  󿬂 owchannel experiments, PS increased the mRNA level of superoxidedismutase (SOD)1, SOD2, catalase, nuclear respiratory factor1 (NRF1), nuclear factor erythroid 2-related factor 2 (Nrf2),Krüppel-like factor (KLF)2, heme oxygenase (HO)-1, and thio-redoxin (Trx)1 in ECs, which are involved in the SIRT1-regulatedantioxidative effect (25 – 30). More importantly, CaMKK  β  knock-down abolished this effect of PS (Fig. 4  A ). To establish thatCaMKK  β  phosphorylation of SIRT1 mediates the induction of these antioxidative and anti-in 󿬂 ammatory genes, we comparedmRNA levels of these genes in SIRT1 −  /  − MEFs transfected withpcDNA control vector, SIRT1-S27AS47A, or SIRT1-S27DS47D.Levels of all these mRNAs were higher in MEFs transfected withSIRT1-S27DS47D than pcDNA or SIRT1-S27AS47A (Fig. 4  B ).In parallel, MEFs transfected with SIRT1-S27DS47D showedincreased SIRT1 activity (Fig. 4 C ). Because SIRT1 deacetylateseNOS with attendant increase in eNOS activity (5), MEFsexpressing SIRT1-S27DS47D also showed enhanced eNOS-derived NO (Fig. 4  D ). CaMKK β  Knockout Increases Oxidative Stress and In 󿬂 ammation in theArterial Wall.  To extrapolate the  󿬁 ndings from  󿬂 ow channel ex-periments to in vivo conditions, we examined whether the levels of total SIRT1 and phosphorylated SIRT1 vary by location in the Fig. 4.  Phosphorylation of SIRT1 Ser-27 and Ser-47 increases SIRT1 activityand expression of SIRT1 target genes. (  A  and  B ) Levels of SOD1, SOD2, cata-lase, NRF1, Nrf2, KLF2, HO-1, and Trx1 mRNA (relative to GAPDH) in HUVECstransfected with control siRNA or CaMKK β  siRNA (  A ) and in SIRT1 −  /  − MEFstransfected with pcDNA, SIRT1-S27AS47A, or SIRT1-S27DS47D ( B ) and thenexposed to PS or static conditions for 8 h. ( C   and  D ) SIRT1 −  /  − MEFs weretransfected with expression plasmids as indicated. Whole-cell lysates werecollected for SIRT1 activity assays ( C  ) and NO bioavailability expressed as NOx( D ). * P   <  0.05. Fig. 5.  CaMKK β  and SIRT1 are involved in the regulation of antioxidativeand anti-in 󿬂 ammatory genes in mouse aorta. (  A ) Immunoblots of tissuelysates from the aortic arch (AA) and the thoracic aorta (TA) isolated fromCaMKK β +/+ mice and their CaMKK β −  /  − littermates performed with indicatedantibodies. Bar graphs to the right summarize the means  ±  SEM from sixmice in each group. ( B ) Levels of SOD1, SOD2, catalase, NRF1, Nrf2, KLF2,PGC1 α , eNOS, ICAM-1, VCAM-1, E-selectin, and MCP-1 mRNA (relative toGADPH) in TA from CaMKK β −  /  − and their CaMKK β +/+ littermates. The resultssummarize the means  ±  SEM from 15 mice in each group. * P   <  0.05. Wen et al. PNAS  |  Published online June 10, 2013  |  E2423       P      H      Y      S      I      O      L      O      G      Y      P      N      A      S      P      L      U      S  arterial tree as a function of   󿬂 ow pattern and, if so, whetherCaMKK  β  is involved. Because mouse SIRT1 has a glutamine atresidue 27 and a serine at residue 46 (homologous to human Ser-47), only phosphorylation at Ser-46 was monitored for the mousestudies. Wild-type, namely CaMKK  β +/+ mice, showed higherlevels of SIRT1, phosphorylated SIRT1 at Ser-46 and phosphor- ylated AMPK at Thr-172 in the thoracic aorta, which is exposed toatheroprotective  󿬂 ow, compared with the aortic arch, which isunder atheroprone  󿬂 ow (Fig. 5  A ). Importantly, CaMKK  β −  /  − lit-termates did not show increased SIRT1 expression or phosphor- ylation of SIRT1 or AMPK in thoracic aortas. In addition, weperformed  en face  staining on the aortic arch and thoracic aortaof CaMKK  β +/+ or CaMKK  β −  /  − mice to compare the expressionlevels of CaMKK  β  and SIRT1. As shown in Fig. S2, the thoracicaorta of CaMKK  β +/+ mice exhibited higher levels of anti-CaMKK  β  and anti-SIRT1 staining than the aortic arch. However,inCaMKK  β −  /  − mice,thestainingforbothanti-CaMKK  β  andanti-SIRT1 was weak and not signi 󿬁 cantly different between thoracicaorta and aortic arch. Taken together, these data indicated thatthe expression of SIRT1 in the arterial tree was regulated by thelocal  󿬂 ow patterns in a CaMKK  β -dependent manner.Consistent with the antioxidative and anti-in 󿬂 ammatory effectsof atheroprotective 󿬂 ow, the mRNA levels of the atheroprotectivegene products SOD1, SOD2, catalase, NRF1, Nrf2, PGC1 α , andeNOS were signi 󿬁 cant lower in the thoracic aorta of CaMKK  β −  /  − mice compared with those in CaMKK  β +/+ mice (Fig. 5  B ). Con- versely, the mRNA levels of intercellular adhesion molecule(ICAM)-1, vascular cell adhesion molecule (VCAM)-1, E-selec-tin, and monocyte chemotactic protein (MCP)-1 were higher inthe thoracic aorta of CaMKK  β −  /  − mice (Fig. 5  B ). Higher levels of ICAM-1, VCAM-1, E-selectin, and MCP-1 were also found in PS-treated ECs in which CaMKK  β  had been knocked down (Fig. S3).Thus, CaMKK  β  ablation led to prooxidative and proin 󿬂 ammatory states in vivo and in vitro. CaMKK β -SIRT1 Is Antiatherogenic in Mouse Arterial Walls. Theresultsin Figs. 1 – 5 indicate that the CaMKK  β -SIRT1 pathway maintainsan antioxidative and anti-in 󿬂 ammatory phenotype of ECs. There-fore, we compared the topographic distribution and severity of atherosclerosis in mice with ablation of CaMKK  β  or SIRT1.CaMKK  β −  /  − mice were crossed with apolipoprotein (Apo)E −  /  − togenerate CaMKK  β −  /  −  /ApoE −  /  − and their CaMKK  β +/+  /ApoE −  /  − littermates. In parallel, EC-SIRT1 +/+  /ApoE −  /  − and EC-SIRT1 −  /  −  /  ApoE −  /  − mouse lines were also obtained. After receiving a Paigendiet for 9 wk, mice were killed and  en face  staining was performedon the aortic specimens. As illustrated in Fig. 6  A  and  B , the totallesion area was 2.2-fold greater in CaMKK  β −  /  −  /ApoE −  /  − thanCaMKK  β +/+  /ApoE −  /  − mice. The lesion areas in the aortic archand thoracic aorta were 12  ±  1.4% and 1.6  ±  0.2%, respectively,for CaMKK  β +/+  /ApoE −  /  − mice and 18  ±  2.8% and 5.7  ±  0.6%,respectively, for CaMKK  β −  /  −  /ApoE −  /  − mice (Fig. 6  B ). Notably,the serum levels of total cholesterol and low-density lipoprotein were similar for the two mouse groups (Table S3). Compared withEC-SIRT1 +/+  /ApoE −  /  − mice, diploid  SIRT1  de 󿬁 ciency in theendothelium caused a 2.4-fold increase in atherosclerosis(Fig. 6 C and  D ). The lesion areas in the aortic arch and thoracic aorta were10 ± 1.9% and 1.5 ± 0.3%, respectively, for EC-SIRT1 +/+  /ApoE −  /  − mice and 21 ± 3.5% and 5.7 ± 1.2%, respectively, for EC-SIRT1 −  /  −  /  ApoE −  /  − mice. Similar to CaMKK  β  knockout, SIRT1 ablationin the endothelium did not alter the lipid pro 󿬁 le (Table S4). Inall, CaMKK  β  ablation in connection with decreased SIRT1 ex-pression in the endothelium increase atherosclerosis in mouseaorta, which suggests the atheroprotective role of   󿬂 ow-inducedCaMKK  β -SIRT1. Discussion The principal  󿬁 ndings of this study are that ( i ) CaMKK  β  phos-phorylates SIRT1 at Ser-27 and Ser-47 in response to atheropro-tective PS in ECs, ( ii ) phosphorylation of SIRT1 at Ser-27 andSer-47 promotes SIRT1 stability and deacetylase activity, and ( iii ) Fig. 6.  CaMKK β  and SIRT1 ablation enhances atherogenesis in mouse aorta. Macrophotographs of oil red O-stained aorta from CaMKK β +/+ ApoE −  /  − andCaMKK β −  /  − ApoE −  /  − mice (  A ) and EC-SIRT1 +/+  /ApoE −  /  − and EC-SIRT1 −  /  −  /ApoE −  /  − mice ( C  ) fed a Paigen diet for 9 wk and killed. (Scale bar: 0.5 cm.) ( B  and  D )Quanti 󿬁 cation of percentage of lesion areas in the whole aorta ( Left  ) and aortic arch (AA) and thoracic aorta (TA) ( Right  ). * P   <  0.05; n denotes the number ofanimals used. E2424  |  www.pnas.org/cgi/doi/10.1073/pnas.1309354110 Wen et al.
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